Download DDT uptake by arbuscular mycorrhizal alfalfa and depletion in soil

DDT uptake by arbuscular mycorrhizal alfalfa and depletion in soil as
influenced by soil application of a non-ionic surfactant
Wu, N. Y., Zhang, S. Z., Huang, H. L., Shan, X. Q., Christie, P., & Wang, Y. S. (2008). DDT uptake by arbuscular
mycorrhizal alfalfa and depletion in soil as influenced by soil application of a non-ionic surfactant. Environmental
Pollution, 151(3), 569-575. DOI: 10.1016/j.envpol.2007.04.005
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Environmental Pollution
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Environmental Pollution 151 (2008) 569e575
www.elsevier.com/locate/envpol
DDT uptake by arbuscular mycorrhizal alfalfa and depletion in soil
as influenced by soil application of a non-ionic surfactant
Naiying Wu a, Shuzhen Zhang a,*, Honglin Huang a, Xiaoquan Shan a,
Peter Christie b, Youshan Wang c
a
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences,
Chinese Academy of Sciences, P.O. Box 2871, Beijing 100085, China
b
Agricultural and Environmental Science Department, Queen’s University Belfast, Newforge Lane, Belfast BT9 5PX, UK
c
Municipal Academy of Agriculture and Forestry, Institute of Plant Nutrition and Resources, Beijing 100097, China
Received 13 November 2006; received in revised form 30 March 2007; accepted 8 April 2007
Combined colonization of alfalfa roots by an arbuscular mycorrhizal fungus and addition of non-ionic surfactant to the soil
promoted root and shoot uptake and soil dissipation of DDT.
Abstract
A greenhouse pot experiment was conducted to investigate the colonization of alfalfa roots by the arbuscular mycorrhizal (AM) fungus
Glomus etunicatum and application of the non-ionic surfactant Triton X-100 on DDT uptake by alfalfa and depletion in soil. Mycorrhizal
colonization led to an increase in the accumulation of DDT in roots but a decrease in shoots. The combination of AM inoculation and Triton
X-100 application enhanced DDT uptake by both the roots and shoots. Application of Triton X-100 gave much lower residual concentrations of
DDT in the bulk soil than in the rhizosphere soil or in the bulk soil without Triton X-100. AM colonization significantly increased bacterial and
fungal counts and dehydrogenase activity in the rhizosphere soil. The combined AM inoculation of plants and soil application of surfactant may
have potential as a biotechnological approach for the decontamination of soil polluted with DDT.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Phytoremediation; Alfalfa; DDT; Glomus etunicatum; Triton X-100
1. Introduction
Contamination of soils by persistent organic pollutants
(POPs) is a widespread environmental problem and their
removal from soil has become a major concern. As a result,
remediation methodologies are of significant interest and considerable attention has been focused on phytoremediation due
to its convenience, cost-effectiveness and environmental
acceptability (Suresh and Ravishankar, 2004).
The organochloride insecticide dichlorodiphenyltrichloroethane (DDT, 1,1,1-trichloro-2,2,bis( p-chlorophenyl)-ethane)
* Corresponding author. Tel.: þ86 10 62849683; fax: þ86 10 62923563.
E-mail address: [email protected] (S. Zhang).
0269-7491/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2007.04.005
was used throughout the world for several decades to control
arthropod disease vectors and agricultural pests. Soil DDT
contamination levels up to 7.5 mg kg1 have been reported
(Kantachote et al., 2004). Although its use in agriculture
was banned in China in 1983, unfortunately, it appears that
its residues and metabolites DDD (1,1-dichloro-2,2-bis( pchlorophenyl)ethane) and DDE (1,1-dichloro-2,2-bis( p-chlorophenyl) ethylene) are still widely distributed in soils. China
still has two production plants producing DDT as an intermediate for dicofol production and control of malaria (Li et al.,
2006). There is a wealth of information demonstrating that
POPs such as DDT have low availability/mobility because of
their sequestration or weathering in soil. Nonetheless, there
have been some greenhouse and field-scale studies of weathered DDT or DDE uptake in a range of plants with accumulation
570
N. Wu et al. / Environmental Pollution 151 (2008) 569e575
concentrations at the mg kg1 level (Lunney et al., 2004; White,
2002; White et al., 2006). Extensive information exists on the
negative effects of DDT and its metabolites on the environment
and on human health (Vieira et al., 2001; Binelli and Provini,
2003).
Arbuscular mycorrhizal (AM) fungi are ubiquitous in terrestrial ecosystems, forming symbiotic associations with roots of
the majority of plant species (Smith and Read, 1997). The ubiquity of AM fungi, their capacity to enhance the tolerance of
host plants to organic contaminants, and their interactions
with soil microorganisms in the rhizosphere have led to recent
studies on their interactions with organic pollutants in the context of phytoremediation (Joner and Leyval, 2003a; Huang
et al., 2007). Research has established that AM fungi can
enhance the growth of several plant species in soil with
high polycyclic aromatic hydrocarbon (PAH) concentrations
(Leyval and Binet, 1998). Experimental evidence has been
obtained for the impact of AM on the uptake and dissipation
of some organic pollutants in soil such as PAHs and atrazine
(Joner and Leyval, 2003b; Huang et al., 2007). These
studies indicated that AM fungi conferred some benefit on phytoremediation of soils contaminated with organic pollutants.
The POPs in soil usually exhibit limited bioavailability to
both microorganisms and plants due to their strong affinity
to the soil matrix, especially soil organic matter (Chen et al.,
2005). Phytoremediation of soils contaminated with POPs is
thus hindered by sorption of the pollutants to soil organic matter and their low aqueous solubility (Boldrin et al., 1993;
Stucki and Alexander, 1987). Solubilization agents such as
surfactants have been added to soil to enhance the release of
POPs from the sorbed phase and, thereby, to increase their
aqueous concentrations and bioavailability (Zhou and Zhu,
2005). Although addition of surfactants has been explored in
the cleanup of contaminated soils (Zhu et al., 2003), there
are few reports of their application in phytoremediation. We
hypothesized that the combined application of AM fungi and
surfactants to be an effective strategy for soil phytoremediation owing to the enhanced mobility of POPs in the soil due
to the surfactants, together with increased degradation and
plant uptake of the POPs as well as improved plant establishment due to mycorrhizal colonization.
In the present study we investigated the effects of AM fungal inoculation and the combined effects of AM fungal inoculation and soil application of the surfactant Triton X-100 on
DDT uptake by alfalfa, dissipation of DDT and its metabolites
and the enzyme activities and microbial populations in a soil
which was artificially contaminated with various levels of
DDT. The aim was to evaluate the potential of AM inoculation
and surfactant application to soil for the phytoremediation of
POPs.
2. Materials and methods
2.1. Experimental design
A pot experiment was conducted in which non-mycorrhizal and mycorrhizal plants with no soil application of surfactant and AM-inoculated plants
grown in soil amended with the non-ionic surfactant Triton X-100 was compared. The soil was spiked with high purity DDT (98% purity, Aldrich Chemical) dissolved in hexane to give application rates of 0.0, 2.5, 5.0 and
10.0 mg kg1 and 10 mL Triton X-100 was added to each pot as
a 0.065 g mL1 solution to reach 0.1% (w/w) in the soil. The treatments
were set up in triplicate.
2.2. Soil preparation
A loamy soil was collected from the surface (0e15 cm depth) of an experimental field at Beijing Academy of Agriculture and Forest Sciences. The selected characteristics are as follows: silt, 45%; clay, 23%; sand, 32%; pH, 7.74
(1:2, soil/water); organic matter, 2.7%; NaHCO3-extractable P, 3.9 mg kg1;
C, 1.95%; N, 0.24%; cation exchange capacity, 25.6 cmol kg1; and an initial
DDT concentration of 0.13 mg kg1. Soil cation exchange capacity was analyzed using the method of Rhodes (1982). Other soil characteristics were determined following the methods described by Srivastava (1992). The soil was
air-dried, ground and passed through a 2-mm nylon sieve. It was then mixed
with sand (1e2 mm) in a ratio of 1:1 (w/w) to produce the growth medium.
The soil mixture (henceforth referred to as the soil) was sterilized by g-radiation (10 kGy, 10 MeV g-rays) to inactivate AM fungi and received mineral
nutrients at rates of 30 mg P (KH2PO4), 60 mg N (NH4NO3), and 67 mg K
(K2SO4) kg1 soil. It was then artificially spiked with DDT with thorough mixing. The soils were allowed to dry in a fume hood for 3 days. During this period the soil samples were shaken three times a day in order to mix the
compounds with the soil thoroughly and to increase the hexane volatilization
rate. Finally, the soils were shaken, homogenized and incubated for 4 weeks at
room temperature. We left a plate in the growth chamber with some resin inside as adsorbent to test the volatilization of DDT.
2.3. Inoculum and host plants
Inoculum of the AM fungus Glomus etunicatum (BGC USA01) was propagated for 10 weeks in pot culture on broomcorn (Sorghum vulgare Pers.)
plants grown in a loamy soil in a greenhouse. The inoculum, which was airdried and passed through a 2-mm sieve, consisted of spores, mycelium, sandy
soil and root fragments containing approximately 350 spores g1 soil (dry
weight soil basis).
Alfalfa seeds (Medicago sativa L.) were purchased from the Chinese Academy of Agricultural Sciences, Beijing, China. They were surface sterilized in
a 10% (v/v) solution of hydrogen peroxide for 10 min, rinsed with sterile distilled water and pre-germinated on moist filter paper overnight and were then
ready for sowing.
2.4. Pot experiment
Each pot received 650 g of incubated soil and was equilibrated in a growth
chamber for 4 d at 70% of water holding capacity. Seven pre-germinated alfalfa seeds were sown in each pot and thinned to four seedlings after growth
for 7 d. Mycorrhizal treatments received 50 g of the fungal inoculum by mixing with about 200 g of soil and then placing the mixture in the middle layer of
the plant pots. In the non-mycorrhizal treatments the inoculum was replaced
by an equivalent amount of sterilized soilesand mixture. For the treatments
with surfactant, Triton X-100 was watered into the soil after sowing to give
a concentration of 0.1% (w/w). The pots were positioned randomly in the
growth chamber and re-randomized every 2 days. The experiment was conducted in a controlled-environment growth chamber with a photoperiod of
14 h at a light intensity of 250 mmol m2 s1 provided by supplementary illumination. The day/night temperature regime was 25 C/20 C and the relative
humidity was maintained at 70%. Distilled water was added as required to
maintain soil moisture content at 70% of water holding capacity by regular
weighing. Nitrogen fertilizer (as NH4NO3) was added to each pot 30 and
45 d after sowing to provide a total of 120 mg N per pot.
N. Wu et al. / Environmental Pollution 151 (2008) 569e575
2.5. Sample preparation
Plants were harvested after growth for 60 d. Pots were left unwatered for 2 d
prior to harvest. Shoots and roots were harvested separately. Any part of the
plant above the soil surface was considered as shoot material and the part below
the soil surface was included in the roots. Root fragments were collected by
sieving the soil and adding them to the root samples. Roots were first carefully
washed with tap water to remove any adhering soil particles. Then roots and
shoots were rinsed thoroughly with distilled water, blotted dry and weighed.
Soils were then sampled from each pot. Bulk soil was collected by gently crushing the soil and shaking the roots. The soil that adhered to the root system was
obtained by shaking the roots and operationally defined as rhizosphere soil
(Lynch, 1990). For enumeration of bacteria and fungi, rhizosphere soil sub-samples were stored at 4 C for microbial assays and were performed within a week.
A portion of fresh root sub-sample was taken from each treatment for the determination of the proportion of root length colonized by the AM fungus. Soil samples and the remainder of the plant samples were freeze-dried, weighed, and
stored at 4 C. Some freeze-dried plant samples were weighed, oven dried at
105 C for 24 h and re-weighed, and the differences between the oven dry
weights and the freeze-dried weights were found to be negligible.
571
with 20 mL hexane and the extracts were concentrated in a rotary evaporator.
The CaCl2 fraction represented DDT dissolved in the aqueous solution within
the apparent free space of the roots. The fraction representing the DDT strongly
adsorbed on the root surface was extracted with 20 mL hexane for 2 h. The hexane solution was then purified using the same method as that used for soils and
plant tissues. Following the hexane extraction, the root tissues were freezedried, ground and subjected to Soxhlet extraction.
Extracts were analyzed using an Agilent 6890 GC equipped with a 63Ni
electron capture detector and a capillary column (30 m 0.25 mm 0.32 mm).
The column oven was programmed from an initial temperature of 80 C to
240 C at a rate of 20 C min1, held for 1 min, and then ramped at a rate of
8 C min1 to 280 C with a final hold time of 1 min. The detector and injector
were maintained at 280 and 220 C, respectively, and the injector was in the
splitless mode. Nitrogen was the carrier gas at 1 mL min1. Injection volume
was 1 mL in n-hexane. Recoveries for the extraction procedure and GC analysis
were tested by spiking a proportion of DDT into the soil and the ground plant
materials which were then incubated for 60 d. The recoveries averaged 90.3%
with an RSD < 4.5% (n ¼ 5).
2.9. Microbial enumeration and determination of enzyme
activities
2.6. Adsorption of DDT on roots
Adsorption of DDT on roots was carried out in batch equilibration sorption
experiments. Three milligram of freeze-dried roots from both inoculated and
uninoculated treatments, from the non-spiked soil, were placed in 50-mL glass
centrifuge tubes containing 35 mL of distilled water saturated with 7 mg L1
DDT. They were then sealed with Teflon-lined screw caps and incubated for
0.5, 1.5, 5, 10, 24, and 48 h on a reciprocating shaker at 25 C. Thirty milliliter
of the sample solutions were taken at different time intervals after centrifuging
for 20 min at 4000 rpm and extracted with 1 mL hexane in preparation for
DDT analysis. The adsorption experiment was carried out in triplicate and
the results were expressed as percentage of DDT adsorbed on roots.
2.7. Assessment of AM colonization of alfalfa roots
The proportion of total root length colonized by the AM fungus was assessed by cutting a random sub-sample of 1 g fresh roots into 0.5- to 1.0cm-long segments. Root segments were cleaned in 10% KOH for 10 min at
90 C in a water bath, rinsed in water, and then stained with 0.1% Trypan
blue for 3e5 min at 90 C in a water bath. Mycorrhizal colonization was determined by the grid line intersect method (Giovannetti and Mosse, 1980).
2.8. DDT analysis
All soil samples were subjected to Soxhlet extraction. One gram of soil
was placed in a cellulose extraction thimble with an equivalent quantity of anhydrous sodium sulphate and extracted with 100 mL of hexane/acetone mixture (1:1, v/v) for 24 h. A cleanup step prior to analysis was mandatory in
order to improve detection and quantification limits and to extend the column
lifetime. The cleanup was done using a modification of the method of Gong
et al. (2004). Briefly, the sample extract was transferred to a separating funnel
and cleaned with 6 mL of concentrated sulfuric acid three times, then cleaned
three times with 30 mL 2% Na2SO4 aqueous solution and dried by passing
through a column packed with 2 g of anhydrous Na2SO4. Finally, it was concentrated and ready for DDT analysis.
The same Soxhlet extraction was conducted on the tissue samples after they
were chopped with pruning shears and ground up using a mortar and pestle, by
which we obtained the contents of DDT inside the roots plus the DDT absorbed
on the root surface. In order to detect the DDT fraction on the root surface, a sequential extraction was carried out before the Soxhlet extraction using a modification of the method of Chen et al. (2007). Three grams of mycorrhizal and
non-mycorrhizal fresh roots from soil spiked with 10 mg kg1 DDT were
washed with distilled water and placed in 50 mL glass centrifuge tubes with Teflon-lined screw caps. They were then extracted with 20 mL of 0.01 mol L1
CaCl2 for 2 h and the bulk solution then filtered. The filtrate was extracted twice
Culturable bacterial and fungal counts were made using the plate count
method. Microbial counts were determined by means of three replicate rhizosphere soil samples. Plate counts of bacteria and fungi were estimated from
106 and 103 dilutions plated on beef extract peptone and Martin’s medium
which were incubated at 30 C for 2 d and 5 d, respectively. After incubation
the numbers of bacterial cells and fungi were counted.
Rhizosphere dehydrogenase activity was assayed using a modification of
the method of Dick et al. (1996). Six grams of rhizosphere soil were thoroughly mixed with 0.06 g CaCO3 and three replicate samples were placed
in test tubes and 3 mL of 1% 2, 3, 5-triphenyltetrazolium chloride (TTC)
and 2.5 mL distilled water were added. Samples were incubated at 37 C for
24 h and then 10 mL methanol added to each tube and the samples were vortexed. The soil suspensions were then filtered and the filtrates were diluted
with methanol to 50 mL. The color’s intensity was measured at 485 nm using
a spectrophotometer. Dehydrogenase activities in the samples were calculated
using calibration curves prepared from 2, 5, 10, and 20 and 30 mg triphenyl
formazan (TPF) mL1 standards. Results are presented as mg TPF g1 soil.
2.10. Data analysis
Data were subjected to two-way analysis of variance using the SPSS version 10.0 software package to determine the significance of soil DDT concentration and AM inoculation with or without the surfactant as sources of
variation. Comparisons between means were carried out using Duncan’s multiple range test at a significance level of p < 0.05.
3. Results
3.1. Root colonization and plant biomass
Table 1 displays the mycorrhizal colonization and biomass
of alfalfa after growth for 60 d. No mycorrhizal colonization
was observed in the roots of uninoculated plants. Mycorrhizal
colonization of the roots of inoculated plants decreased from
70.3 to 49.7% with increasing DDT concentration from 0 to
10 mg kg1. Mycorrhizal root colonization varied from 66.2
to 49.1% for the treatments that included Triton X-100. Mycorrhizal plants had higher shoot yields and lower root yields
than non-mycorrhizal controls with only one exception when
DDT was applied at 10 mg kg1. DDT application generally
increased shoot and root biomass and application of Triton
N. Wu et al. / Environmental Pollution 151 (2008) 569e575
572
Table 1
Mean shoot and root dry matter yield (freeze-dried basis) and proportion of root length colonized by the AM fungus after cultivation of mycorrhizal alfalfa with or
without surfactant and non-mycorrhizal controls in soil containing different levels of added DDTa (mean SE, n ¼ 3)
Initial DDT
addition (mg kg1)
Mycorrhizal status/surfactant
addition
Shoot weight
(g pot1)
Root weight
(g pot1)
Mycorrhizal colonizationb
(%)
0.0
Non-mycorrhizal
Mycorrhizal
Mycorrhiza þ surfactant
0.91 0.04g
1.16 0.07bd
0.82 0.03gh
0.70 0.03c
0.65 0.06fh
0.56 0.01g
0.0
70.3 1.29a
66.2 0.83b
2.5
Non-mycorrhizal
Mycorrhizal
Mycorrhiza þ surfactant
1.16 0.09bcdef
1.29 0.01abde
1.05 0.05efg
0.74 0.10d
0.66 0.09eh
0.62 0.02bcdefgh
0.0
68.5 0.57a
59.2 3.03c
5.0
Non-mycorrhizal
Mycorrhizal
Mycorrhiza þ surfactant
1.14 0.12de
1.34 0.11ac
1.37 0.08ab
1.06 0.06a
0.63 0.09f
0.84 0.13b
0.0
52.7 2.52d
51.3 3.27d
10.0
Non-mycorrhizal
Mycorrhizal
Mycorrhiza þ surfactant
1.23 0.04abdef
1.39 0.04a
1.00 0.05fh
0.82 0.03bcd
0.85 0.06abcde
0.79 0.06c
0.0
49.5 2.53d
49.2 1.22d
***
***
*
***
***
***
***
***
***
Significance of
DDT level
Mycorrhizal status/surfactant
DDT mycorrhizal status/surfactant
*Significant by analysis of variance at p < 0.05.
***Significant by analysis of variance at p < 0.001.
a
Values followed by the same letter within a column are not significantly different according to Duncan’s multiple range test at the 5% level.
b
Colonization of NM pants was 0%, it was therefore excluded from statistical analysis of effects of DDT/surfactant on mycorrhizal colonization.
X-100 decreased them. Although these yield effects were statistically significant they were numerically small.
3.2. Accumulation of DDT in plants
DDT accumulation in alfalfa is shown in Fig. 1. Roots accumulated much more DDT than did shoots. DDT concentrations
in both shoots and roots increased markedly with increasing
DDT application rate to soil irrespective of inoculation treatment. AM inoculated roots accumulated consistently more
DDT than non-mycorrhizal roots. Addition of Triton X-100
led to increased DDT concentrations in roots. Mycorrhizal
plants showed lower shoot DDT accumulation than non-mycorrhizal controls, but application of Triton X-100 to the soil significantly elevated DDT accumulation in the shoots ( p < 0.05).
rhizosphere soils as well as in bulk soils without Triton
X-100 application. For instance, when 10.0 mg kg1 of DDT
was added to the soil, the DDT concentration decreased to
27.3% of the original level in the bulk soil compared to
87.8% in the bulk soil in the absence of Triton X-100 and
47.3% in the rhizosphere soil in the presence of Triton X-100.
3.4. DDT adsorption on roots
DDT sorption on mycorrhizal and non-mycorrhizal roots
increased rapidly during the first 5 min and then remained constant (Fig. 3). A maximum of up to 79.6% of DDT was sorbed
on mycorrhizal roots compared to 80.3% on non-mycorrhizal
roots. DDT sorption on non-mycorrhizal and on mycorrhizal
roots was not significantly different ( p > 0.05).
3.3. Dissipation of DDT in soils
3.5. Microbial assays and dehydrogenase activities
Negligible changes in the DDT concentration in the spiked
soils were detected after aging for 4 weeks. After harvest,
only DDT was detected in the bulk soil samples but the two metabolites DDD and DDE were detected in the rhizosphere soils
from all treatments (Fig. 2). However, the concentrations were
very low and no clear differences in concentrations could be detected among the different treatments. The DDT concentrations
in soil decreased markedly compared with the initial levels and
this effect was more pronounced in the rhizosphere soil than in
the bulk soil. A 66.8e95.4% reduction of the initially added
DDT was observed in the rhizosphere soils compared to
13.1e64.8% in the bulk soils. An interesting observation was
that in the presence of Triton X-100 the DDT residual concentrations were much lower in the bulk soils than in the
Soil microbial counts and dehydrogenase activities in rhizosphere soil varied among the treatments (Table 2). Total culturable bacteria and fungi varied from 1.0 107 to 3.4 107 cells g1 and 2.8 107 to 4.4 107 cells g1. The mean
bacterial numbers were at least three orders of magnitude higher
than the fungal counts. AM inoculation significantly increased
bacterial and fungal counts. Dehydrogenase activity changed
in a similar way to the counts of soil microorganisms.
4. Discussion
A major finding of the present study is the impact that AM
fungal colonization and Triton X-100 had on the accumulation
of DDT in alfalfa roots and shoots. We hypothesized that such
N. Wu et al. / Environmental Pollution 151 (2008) 569e575
a
a
DDT in shoots (mg kg-1)
3
b
2
c
1
d
e
f
g
d
d
f
e
2.5
5
0
0
10
DDT added to soil (mg kg-1)
250
b
DDT in roots (mg kg-1)
200
a
150
100
c
e
f
50
g
l
j
b
d
h
i
k
0
0
2.5
5
10
DDT added to soil (mg kg-1)
Fig. 1. DDT concentrations in alfalfa (a) shoot and (b) root tissues by Soxhlet
extraction. Data are means of three replicates and on a dry matter (freezedried) basis. Non-mycorrhizal (
); Mycorrhizal (
); Mycorrhiza þ surfactant (
). Bars, standard errors. Values followed by the same letter are not significantly different according to Duncan’s multiple range test at
the 5% level.
an observation might be attributable mainly to an increase in
the adsorption of DDT on colonized roots. In order to test
this hypothesis we examined DDT sorption by excised root tissues and found that DDT adsorption was consistently higher
on non-mycorrhizal than on mycorrhizal roots. There was no
significant difference between mycorrhizal and non-mycorrhizal plants at the 5% level (Fig. 3). The adsorption experiment
had some limitations due to the low solubility of DDT in water
and it was not possible to elucidate the sorption behavior of
DDT at high concentrations by experiment or by isotherm
model prediction. We therefore proceeded to use a sequential
extraction procedure with CaCl2 and hexane to extract DDT
from the fresh roots harvested from the soil spiked with
10 mg kg1 DDT before Soxhlet extraction in order to clarify
the impact of AM fungal colonization on the affinity of roots
for DDT. The CaCl2 extract fraction, which was considered as
loosely adsorbed on root surfaces, was too low to be detected.
573
The hexane fraction (strongly adsorbed fraction) of DDT accounted for 5.6% and 17.2% of the total amount of DDT in
mycorrhizal and non-mycorrhizal roots, respectively. This result confirms that sorption and sequestration of DDT was
higher on mycorrhizal roots than on non-mycorrhizal roots
and it might be ascribed to the formation of abundant extraradical mycelium (Joner et al., 2001) which has a high affinity
with hydrophobic organic compounds. The combination of
mycorrhizal inoculation and the surfactant Triton X-100 increased DDT accumulation in both roots and shoots. Fig. 1
shows much greater DDT accumulation in the roots in the
presence of Triton X-100, especially at the highest DDT
dose rate studied, which may have contributed to the increase
in DDT translocation to the shoots. Another possible explanation for the enhanced accumulation of DDT in mycorrhizal
roots could be an increase in the mobility and bioavailability
of the contaminant in soil. It is possible that the AM fungus
stimulated the mineralization of DDT in the soil via its effects
on soil organisms as can be seen from the difference in bacterial counts, which in turn may have led to the changes in DDT
availability. However, elucidation of any direct effect needs to
be addressed by further work.
After plants’ harvest the total residual concentrations of
DDT in soil decreased significantly (Fig. 2). The trends following addition of Triton X-100 to the soil are of particular
interest. The non-ionic surfactant greatly decreased the residual concentrations of DDT in the bulk soil while increasing
the concentrations in the rhizosphere soil. Earlier studies
(Kile and Chiou, 1989) have found that surfactants such as
the Triton series and Brij35 significantly enhanced DDT solubility in soils owing to micelle formation. Increased solubility of DDT in soil as well as enhanced plant uptake of water
facilitated by AM colonization (Morales Vela et al., 2007)
may promote mass flow of DDT towards roots and sequestration in the rhizosphere, a soil zone which may promote
DDT degradation and may also contain ample sites for adsorption of POPs on organic matter. Furthermore, formation
of abundant extraradical mycelium by AM colonization can
also benefit the sequestration of DDT in the rhizosphere
zone. The rhizosphere therefore acts as a sink for DDT.
This could be very important in phytoremediation because
sequestration of POPs in the soil and limited bioavailability
are important obstacles in the application of bioremediation
methods.
Phytoremediation of organic pollutants depends on plante
microbe interactions in the rhizosphere (Joner and Leyval,
2003b). We observed a pronounced influence of the AM fungus
on soil microbial abundance. It is now well established that AM
fungi modify root functions (i.e. root exudation), change carbohydrate metabolism of the host plant and influence rhizosphere
populations (Duponnois et al., 2005). Furthermore, AM fungi
can exude substances that have a selective effect on the microbial community in rhizosphere soil and thus modify the microbial communities. Previous studies (Stelmack et al., 1999; Chen
et al., 2000) have demonstrated inhibitory effects of non-ionic
surfactants on bacterial growth, even under the critical micelle
concentration. However, we did not find any significant
N. Wu et al. / Environmental Pollution 151 (2008) 569e575
5
a
82
a
DDT
DDD
DDE
M
NM
4
b
3
d
f
DDT adsorbed on roots (%)
DDT and its metabolites concentration
(mg kg-1)
574
bc
c
2
1
e
gg
h
81
80
79
78
0
0
2.5
5
10
DDT added to soil (mg kg-1)
77
DDT concentration (mg kg-1)
10
0
b
20
10
a
8
30
40
50
t (h)
Fig. 3. DDT adsorbed on alfalfa roots. Results are expressed as percentage of
DDT adsorbed on roots (n ¼ 3). Bars, standard errors.
b
6
difference in the microbial counts ( p > 0.05) between the mycorrhizal and mycorrhizal plus surfactant treatments. Negative
effects of surfactant application might be alleviated by AM
colonization.
c
4
d
e
2
h
i i
f
f
5. Conclusions
g
i
0
0
2.5
5
To our knowledge, the present study is the first to report the
effects of arbuscular mycorrhizal colonization combined with
application of surfactant to the soil on DDT uptake by plants
and DDT dissipation in the soil. AM colonization assisted in
the uptake of DDT by alfalfa roots and the combined effects
of AM colonization and Triton X-100 increased the accumulation of DDT in both the roots and shoots. In addition, Triton
10
DDT added to soil (mg kg-1)
Fig. 2. Total DDT concentrations in (a) rhizosphere and (b) bulk soil. Data are
means of three replicates. Non-mycorrhizal (
); Mycorrhizal (
); My). Bars, standard errors. Values followed by the
corrhiza þ surfactant (
same letter are not significantly different according to Duncan’s multiple range
test at the 5% level.
Table 2
Mean bacterial and fungal counts (CFU/g) and soil dehydrogenase activity in rhizosphere soila (mean SE, n ¼ 3)
Initial DDT addition (mg kg1)
Mycorrhizal status/surfactant addition
0.0
Non-mycorrhizal
Mycorrhizal
Mycorrhiza þ surfactant
2.5
Bacterial counts (107)
Fungal counts (104)
15.3 0.80i
47.5 1.30h
14.8 0.30i
1.42 0.09f
2.41 0.15cd
2.22 0.07d
3.64 0.21cd
4.20 0.28abc
3.92 0.14c
Non-mycorrhizal
Mycorrhizal
Mycorrhiza þ surfactant
200.7 2.14f
216.0 10.23df
78.1 2.73g
1.75 0.04e
2.46 0.1cd
2.31 0.07cd
3.93 0.13c
4.64 0.18a
4.17 0.17ac
5.0
Non-mycorrhizal
Mycorrhizal
Mycorrhiza þ surfactant
216.1 6.06d
323.2 4.79a
169.9 2.91e
2.30 0.22cd
2.87 0.05b
2.50 0.43bcd
3.10 0.36de
4.43 0.13ab
4.0 0.23bc
10.0
Non-mycorrhizal
Mycorrhizal
Mycorrhiza þ surfactant
277.4 5.65c
307.8 5.12b
215.9 3.56d
2.62 0.19bc
3.41 0.15a
3.00 0.16ab
2.67 0.09e
3.25 0.19d
2.83 0.1e
***
***
***
***
***
n.s.
***
***
n.s.
Significance of
DDT level
Mycorrhizal status/surfactant
DDT mycorrhizal status/surfactant
Dehydrogenase (mg/kg/24 h)
***Significant by analysis of variance at p < 0.001.
n.s., Not significant.
a
Values followed by the same letter within a column are not significantly different according to Duncan’s multiple range test at the 5% level.
N. Wu et al. / Environmental Pollution 151 (2008) 569e575
X-100 increased the mobility and thereby the transport of DDT
from the bulk soil to the rhizosphere, an effect which may have
promoted both plant uptake and soil degradation of DDT. The
combined use of AM inoculum and surfactants may have
some potential as a biotechnological approach for the
decontamination of soils contaminated with organic pollutants.
However, detailed and comprehensive studies on the selection of
plant species, AM fungi and surfactants will be required to
effective remediation methods under field conditions.
Acknowledgements
This work was funded by the National Natural Science
Foundation of China (Project 20677072, 20621703) and the
Hi-tech Research and Development Program of China (Project
2006AA06Z349).
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